Human Acute Myeloid Leukemia Xenograft Models Research Articles

Synergistic effects of RPT1G, a first-in-class small molecule NAMPT inhibitor, with venetoclax and olaparib in hematological malignancies.

e18512 Background: MultipleBCL-2 and PARP inhibitors are currently in clinical trials, or have been approved for use, in hematological malignancies and solid tumors. Venetoclax, an inhibitor of the apoptosis regulator B-cell lymphoma-2 (BCL-2), has activity in patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). PARP inhibitors, such as Olaparib, exploit synthetic lethality in homologous recombination-deficient tumors, notably in breast and ovarian cancers. However, patients treated with BCL-2 or PARP inhibitors often face relapse and refractory disease, requiring alternative strategies to counter adaptive responses. Nicotinamide Phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, is upregulated in malignancies and plays a key role in cancer metabolism (e.g., Warburg effect). NAMPT makes nicotinamide mononucleotide which is then converted to NAD+, while PARP enzymes consume NAD+, placing NAMPT directly upstream of PARP. NAMPT overexpression is associated with Venetoclax resistance in leukemia patients, highlighting the link between NAD+, mitochondrial metabolism, and BCL-2 inhibition. Consequently, NAMPT inhibition may synergize with BCL-2 or PARP inhibitors, potentially enhancing 1st-line of treatment outcomes and re-sensitizing refractory cancer cells to BCL-2 or PARP inhibition. Methods: In this study, we investigated the combination effects of Venetoclax and Olaparib with RPT1G, a novel hyperbolic NAMPT inhibitor that simultaneously inhibits and stabilizes NAMPT in a catalytically active state. RPT1G disrupts NAMPT activity without ever turning it off completely avoiding the severe on-target toxicities seen with complete NAMPT inhibitors. Several cell lines, including OCI-AML3 and MV4;11 (AML), RS4;11 (ALL), and RL (diffuse large B-cell lymphoma) were treated with RPT1G and either Venetoclax or Olaparib to determine the degree of drug interactions. In a follow up in vivo study, we treated mice in a human xenograft model using semi-resistant MV4;11 cells with RPT1G alone or in combination with Venetoclax to measure the ability of RPT1G to enhance the effects of Venetoclax. Results: RPT1G achieves significant single-agent efficacy in leukemia and lymphoma mouse xenograft models. When RTP1G is used in combination with either Venetoclax or Olaparib significant synergy is seen across a broad range of concentrations in vitro. Consistent with in vitro synergy results, in a xenograft model of human AML, mice treated with a combination of RPT1G and Venetoclax show enhanced efficacy as compared to Venetoclax alone. Conclusions: Combination of RPT1G with either Venetoclax or Olaparib synergistically enhances killing of leukemic cells and presents as an effective strategy to improve treatment outcomes for patients with diverse malignancies.

Pre-Clinical Investigation of a Novel Small Molecule Inhibitor Targeting YBX1 in AML

Recently, our group identified the pleiotropic DNA and RNA binding protein YBX1 as a specific dependency and therapeutic target in acute myeloid leukemia (AML) that is essential for stabilizing oncogenic transcripts and thereby maintains oncogenic protein expression (Perner et al., 2021). Selective pharmacologic targeting of YBX1 in AML, however, has not been achieved so far. Here, we investigate the functional consequences of a novel small molecule inhibitor (SU056) of YBX1 in AML. We examined the viability of several human AML cell lines (MOLM13, HL60, EOL1, THP1, Kasumi1, ML2, Mv4;11, OCI-AML3, NOMO1) upon SU056 treatment and found significant reduction of cell growth in a concentration dependent manner. Inhibition of cell growth could be mainly attributed to impaired proliferative capacity at nanomolar concentrations. Pharmacologic inhibition of YBX1 resulted in impaired colony formation (mean colony number DMSO: 231.3±13.88 vs. SU056 500nM: 154.7±8.29; p<0.0001), cell cycle arrest (cells in S phase: DMSO: 37.3±2.4 vs. SU056 500nM: 27.2±1.67; p=0.0127) and induction of myeloid differentiation. Consistently, in a xenograft model of human AML, SU056 treatment resulted in prolonged disease latency (MOLM13 diluent: 62 days OS vs. SU056: not reached, p=0.0013; OCI-AML3 diluent: 79 days OS vs. not reached, p=0.0001) and significant reduction of disease penetrance (MOLM13 diluent: 100% vs. SU056: 24,9%; OCI-AML3 diluent: 100% vs. SU056: 9,1%). Consistently, percentage of SU056 treated human leukemic cells in the bone marrow of recipient mice was decreased for both, MOLM13 (mean diluent: 41,3% vs. SU056: 7,6%) and OCI-AML3 (mean diluent: 28,9% vs. SU056: 2,9%). Likewise, pharmacologic inhibition of YBX1 reduced colony formation and total cell numbers of primary human AML cells in a dose-dependent manner. To be clinically relevant, such treatment must have a suitable therapeutic index between human hematopoietic stem- and progenitor cells (HSPCs) and their malignant counterpart. This is particularly important, considering the role of YBX1 in mRNA splicing and processing. Of note, pharmacologic inhibition of YBX1 did not perturb colony formation of normal HSPCs derived from apheresis donors. These findings were confirmed by investigating primary human leukemic cells in a patient-derived xenograft (PDX) model of AML. We aimed to assess whether loss in leukemia cell competition induced by pharmacologic targeting of YBX1 is sufficient for normal hematopoietic cells to outcompete leukemic cells in vivo. 1x106 normal human HSPCs were transplanted along with 2x104 leukemic cells to assess for efficacy of SU056-treatment versus diluent control. In vivo treatment with SU056 (20mg/kg, i.p.) for 3 weeks resulted in a significant decrease in the AML blast number in the PB (mean blast %: diluent: 38.5%; SU056: 2.25%; p<0.0001). This resulted in increased survival of SU056 treated recipients (OS diluent: 83 days vs. SU056: not reached). To confirm the proposed mechanism by which SU056 impairs leukemia growth we performed global transcriptome and proteome analysis following treatment with SU056 (500nM, 96h). Proteome profiling revealed a total of 2641 deregulated proteins. Gene-ontology analysis indicated deregulation of YBX1 targets and proteins associated with cell cycle, DNA repair, mRNA splicing/processing, and transcriptional regulation. Of note, overlap between transcriptional regulation (as determined by mRNA-Seq) and changes in proteome abundance revealed less than 5% target genes/proteins, indicating that SU056 may in fact interfere with binding of oncogenic mRNAs to YBX1 and thereby may reduce translation of oncogenic transcripts. In order to define functional consequences of pharmacologic YBX1 inhibition and explore potential vulnerabilities of drug combinations, we performed a genome-wide CRISPR/Cas9 screen in human AML cells treated with SU056 versus diluent control. Validation experiments are currently ongoing. Taken together, our data confirm the therapeutic potential to target YBX1 using the novel small molecule SU056. Given its broad applicability across genetic subtypes of AML and lack of toxicity on normal HSPCs, SU056 as a single agent or in combination with established targeted therapies may efficiently help to target and outcompete leukemic cells. Experiments to assess for potential synergy with already established targeted therapies will be presented.

CKS1 inhibition depletes leukemic stem cells and protects healthy hematopoietic stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low two-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs, and the significant toxicity towards healthy hematopoietic cells. Whilst much work has been done to identify genetic and epigenetic vulnerabilities in AML LSCs, little is known about protein homeostasis in drug resistance and relapse. By targeting the proteostatic regulator CKS1, we demonstrate a dual role for CKS1-dependent protein degradation in reducing AML blasts in vivo, and importantly depleting LSCs, whilst inhibition of CKS1 has the opposite effect on normal hematopoiesis, protecting normal hematopoietic stem cells from chemotherapeutic toxicity. Together these findings demonstrate CKS1-dependent proteostasis is a key vulnerability in malignant stem cell biology.

In This Issue

In This Issue

An In Vivo CRISPR Screening Platform to Identify New Therapeutic Targets in AML

First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing.We first defined experimental conditions necessary for an optimal in vivo screen via barcoding experiments. We determined that sub-lethal irradiation was necessary for improved barcode representation in bone marrow and to reduce mouse-to-mouse variation. Moreover, it was critical to combine samples from multiple mice to achieve complete in vivo library representation. Next, using the Broad DepMap and other publicly available functional genomic screen data, we identified 200 genes that were stronger dependencies in AML cell lines compared to all other cancer types screened. Using this list, we created a secondary library and performed parallel in vivo and in vitro screens using the MV4-11 and U937 cell lines and a PDX model. In vitro and in vivo hits were surprisingly well correlated, although a modest number of targets did not score well in vivo. Notably, dependencies identified across AML cell line models were strongly recapitulated in the PDX model, validating the application of AML cell lines for dependency discovery.Our in vivo screens nominated the mitochondria-localized RING-type ubiquitin E3 ligase MARCH5 as a potential therapeutic target in AML. Using CRISPR, we first validated this in vitro dependency on MARCH5 and determined that MARCH5 is a critical guardian to prevent apoptosis in AML. MARCH5 depletion activates the canonical mitochondrial apoptosis pathway in a BAX/BAK1-dependent manner. Multiple genome-wide screens revealed that a dependency on MARCH5 is strongly correlated with a dependency on MCL1, but not other anti-apoptotic BCL2-family members, across the AML cell lines in the screen. As observed with MCL1 inhibition, MARCH5 depletion sensitized AML cells to venetoclax, a BCL2-specific inhibitor FDA-approved in combination with a hypomethylating agent for the treatment of older adults with AML. Importantly, MARCH5 depletion diminished the venetoclax resistance induced by MCL1 overexpression but not that caused by BCLXL overexpression. Altogether, these results suggest that MARCH5 is required for maintaining MCL1 activity specifically.Since there are no small molecule inhibitors directed against MARCH5, we deployed a dTAG system as an approximation of pharmacological inhibition. This approach uses a hetero-bifunctional small molecule that binds the FKBP12 F36V-fused MARCH5 and the E3 ligase VHL, leading to the ubiquitination and proteasome-mediated degradation of the fusion protein. dTAG-MARCH5 cells were established via deleting endogenous MARCH5 by CRISPR and expressing exogenous FKBP-tagged MARCH5 protein. MARCH5 degradation with the dTAG molecule dTAG V-1 markedly impaired cell growth in vitro. Additionally, we demonstrated the utility of dTAG system in vivo using a PDX model. The combination treatment of dTAG V-1 and venetoclax elicited a much stronger anti-leukemic effect compared to the treatment with only venetoclax or dTAG V-1, further highlighting MARCH5 as a promising synergistic target for enhancing the efficacy of venetoclax in AML.Taken together, our in vivo screening approach, coupled with CRISPR-competent PDX models and dTAG-directed protein degradation, constitute a useful platform for prioritizing AML targets emerging from in vitro screens to serve as the starting point for therapy development. DisclosuresDharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: Bristol Myers Squibb: Consultancy; KronosBio: Consultancy; AstraZeneca: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

Nuclear NAD+ homeostasis governed by NMNAT1 prevents apoptosis of acute myeloid leukemia stem cells.

Metabolic dysregulation underlies malignant phenotypes attributed to cancer stem cells, such as unlimited proliferation and differentiation blockade. Here, we demonstrate that NAD+ metabolism enables acute myeloid leukemia (AML) to evade apoptosis, another hallmark of cancer stem cells. We integrated whole-genome CRISPR screening and pan-cancer genetic dependency mapping to identify NAMPT and NMNAT1 as AML dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, the presence of NAD+ precursors bypassed the dependence of AML on NAMPT but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. Deletion of NMNAT1 reduced nuclear NAD+, activated p53, and increased venetoclax sensitivity. Conversely, increased NAD+ biosynthesis promoted venetoclax resistance. Unlike leukemia stem cells (LSCs) in both murine and human AML xenograft models, NMNAT1 was dispensable for hematopoietic stem cells and hematopoiesis. Our findings identify NMNAT1 as a previously unidentified therapeutic target that maintains NAD+ for AML progression and chemoresistance.

Niche Regulation of Residual Hematopoietic Stem Cell Protein Synthesis By Extracellular Vesicle (EV) Trafficking in the Acute Myeloid Leukemia (AML) Microenvironment

Work by several groups has shown that the presence of Acute Myeloid Leukemia (AML) deregulates critical functions of the bone marrow microenvironment, including hematopoiesis, in part through indirect stroma-secreted factors. We recently reported that Extracellular Vesicles (EV) released from AML blasts directly suppress the clonogenicity of hematopoietic progenitors via the action of miRNA-150 and miR-155 on cMyb, a transcription factor highly expressed in differentiating progenitors. The fate of long-term hematopoietic stem cells (LT- HSC) in the AML niche has not been widely studied. Using a xenograft model of human AML (Molm-14, U937 and HL-60 cell lines), we first ascertained the in vivo uptake of AML-EVs and found different internalization efficiencies among hematopoietic stem and progenitor cell (HSPC) populations, including LT-HSC (CD150+CD48−KSL) by imaging and flow cytometry. Live-cell microscopy studies of LT-HSCs revealed AML-EV binding and uptake within minutes following in vitro exposure. To determine the functional implications of EV trafficking, we next evaluated the relative frequency of the hematopoietic populations in low chimerism xenografts and found a consistent increase in the LT-HSC population, but not multi-potent progenitors, suggestingthe proportional accumulation of LT-HSC population caused by mobilization and lack of expansion and differentiation of other hematopoietic cells. To gain insight into how AML-EVs may regulate cell fate and function following internalization, we performed a proteomic profiling of HSPC cells exposed to EVs from two cell lines representing different AML subtypes. Bioinformatics analysis found significant enrichment for the ribosomal biogenesis pathways and negative cell cycle regulators in the EV-exposed cells. Because LT-HSC have been shown to be particularly susceptible to impairment of ribosome biogenesis, we wished to determine if AML-EVs modulate the LT-HSC fate by suppressing protein synthesis. We therefore injected AML-EVs into the femurs of both immuno-competent and immuno-compromised mice and evaluated the cellular protein synthesis by quantifying O-Propargyl-Puromycin (OPP) incorporation. Results show that AML-EVs significantly suppress the protein synthesis in the LT-HSC, but not in the HSPC pool population. The mTOR signaling pathway is known to be greatly involved in ribosome biogenesis via phosphorylation of the ribosomal protein S6 (S6RP) which in turn regulates several repressors of rRNA and ribosomal proteins. Consistent with the notion of translational suppression, we found that AML-EVs reduced the phosphorylation of S6RP in LT-HSC. In correlation with the observation that ribosomal stress induction via suppression of ribosomal proteins we found upregulation of P53 and its transcriptional target p21, as well as a gain in quiescence (G0) in the LT-HSC population. To address the functional fate of deregulated protein synthesis and quiescence, we transplanted KSL cells purified from male mice engrafted with AML cells into irradiated female recipient mice and measured the donor chimerism via qPCR for Sex-determining Region Y (SRY). Early repopulation capacities between cohorts of mice transplanted with KSL from AML xenograft versus those from control non-engrafted mice are similar (with long-term evaluation ongoing) in the host non-leukemic microenvironment. This suggesting that hematopoietic impairment by AML-EVs is reversible upon transplantation to a different host environment. Collectively, our results provide the first evidence that AML-EV impair LT-HSC via suppressing of ribosomal biogenesis. Understanding the regulation of LT-HSC by AML-EV will help reduce the morbidity and mortality from hematopoietic suppression in patients with leukemia. DisclosuresNo relevant conflicts of interest to declare.

TAS4464, a NEDD8-activating enzyme inhibitor, activates both intrinsic and extrinsic apoptotic pathways via c-Myc-mediated regulation in acute myeloid leukemia

TAS4464, a potent, selective small molecule NEDD8-activating enzyme (NAE) inhibitor, leads to inactivation of cullin-RING E3 ubiquitin ligases (CRLs) and consequent accumulations of its substrate proteins. Here, we investigated the antitumor properties and action mechanism of TAS4464 in acute myeloid leukemia (AML). TAS4464 induced apoptotic cell death in various AML cell lines. TAS4464 treatments resulted in the activation of both the caspase-9-mediated intrinsic apoptotic pathway and caspase-8-mediated extrinsic apoptotic pathway in AML cells; combined treatment with inhibitors of these caspases markedly diminished TAS4464-induced apoptosis. In each apoptotic pathway, TAS4464 induced the mRNA transcription of the intrinsic proapoptotic factor NOXA and decreased that of the extrinsic antiapoptotic factor c-FLIP. RNA-sequencing analysis showed that the signaling pathway of the CRL substrate c-Myc was enriched after TAS4464 treatment. Chromatin immunoprecipitation (ChIP) assay revealed that TAS4464-induced c-Myc bound to the PMAIP1 (encoding NOXA) and CFLAR (encoding c-FLIP) promoter regions, and siRNA-mediated c-Myc knockdown neutralized both TAS4464-mediated NOXA induction and c-FLIP downregulation. TAS4464 activated both caspase-8 and caspase-9 along with an increase in NOXA and a decrease in c-FLIP, resulting in complete tumor remission in a human AML xenograft model. These findings suggest that NAE inhibition leads to anti-AML activity via a novel c-Myc-dependent apoptosis induction mechanism.

Nicotinamide Phosphoribosyltransferase Inhibitors Induce Apoptosis of AML Stem Cells through Dysregulation of Lipid Metabolism

Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents &amp; Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

AML-337: Targeting E-Selectin with GMI-1271 Overcomes Microenvironment-Mediated Resistance to Venetoclax/HMA Therapy

Acute myeloid leukemia (AML) is an aggressive heterogeneous hematologic disease with high mortality in patients older than 60 years. Clinical studies have proven that combinations of FDA-approved Bcl-2 inhibitor, venetoclax and hypomethylating agents (HMA) are highly effective in elderly patients with AML (DiNardo et al., 2019). Adhesion to the BM niche is critical for AML initiation, progression and LSC survival after induction therapy. The vascular adhesion molecule, E-selectin (E-sel) has crucial roles in BM homing and engraftment in leukemia (Krause et al, 2006). Here we elucidated the roles of E-sel in AML survival using human AML cell lines and patient-derived AML xenograft (PDX) models. In our experiments, E-sel decreased CDK4/6 expression and increased dormancy of AML cells in vitro. Pharmacological inhibition of E-sel by GMI-1271 increased expression of cell cycle regulating proteins including CDK4/6 and CyclinD1/2 in HUVEC co-cultured AML. To determine if E-sel has indispensable effects in BM niche component cells, we exposed healthy donor derived-mesenchymal stroma cells (MSC) to increasing concentrations of E-sel: E-sel upregulated surface expression of the most potent E-sel ligand, CD44 in human MSC and abrogation of E-sel binding by GMI-1271 diminished CD44 expression in vitro. Targeting E-sel with GMI-1271 attenuated eNOS phosphorylation in HUVEC co-cultured with AML cells, suggesting that E-sel inhibition may protect disruption of BM vasculatures during AML progression. Further, we found that targeting E-sel mobilized human AML cells and sensitized them to venetoclax/HMA in a PDX model derived from a patient who had developed resistance to venetoclax/HMA. The number of circulating leukemic cells was significantly reduced by combinatorial treatment of GMI-1271 with venetoclax/HMA compared to venetoclax/HMA alone (p<0.05). In addition, the combination of GMI-1271 and venetoclax/HMA significantly prolonged the survival of mice compared to vehicle control (p=0.015) as well as the venetoclax/HMA (p=0.0009) and GMI-1271 groups (p=0.03). The median survival of the vehicle control, GMI-1271, venetoclax/HMA, and combination-treated groups of mice was 86, 91, 81.5, and 106.5 days, respectively. Collectively, our results provide first evidence that an E-sel targeting strategy with GMI-1271 may overcome microenvironmental resistance to venetoclax/HMA-based therapy in AML by disrupting signaling pathways in the BM vascular niche.

Targeting AML: The Development of Two Functionally and Mechanistically Distinct Classes of Cereblon-Mediated Protein Homeostatic Modulators

Targeting disease-relevant proteins by exploiting the cells' very own protein homeostasis machinery is the next generation drug discovery platform that has come center stage for treatment of hematological malignancies. We present our novel and unique Protein Homeostatic Modulators (PHMsTM) - that promote ubiquitination and subsequent proteasomal degradation of known substrates as well as neosubstrates via Cereblon - as therapeutic candidates for acute myeloid leukemia (AML). Specifically, we report the discovery of two functionally and mechanistically distinct classes of Cereblon-mediated PHMsTM from phenotypic screens of our proprietary PHM®library. Unlike classical IMiDs, our oncology candidates, BTX508 and BTX1119 demonstrate significant cytotoxicity with IC50s in low-nanomolar range in a panel of AML cell lines. BTX508 is a purely cytotoxic compound that does not display immune modulatory activity. In contrast, BTX1119 is not only cytotoxic, but is also immunomodulatory in that it inhibits inflammatory cytokines such as IL-1β, IL-6 and TNF-α as well as induces IL-2, an indicator of T cell activation. BTX1119's immune modulatory activity is a 100-fold more potent when compared to Pomalidomide. The distinct functional activities exhibited by BTX508 and BTX1119 is a result of strategically engineered substrate degradation profiles. Importantly, BTX508 and BTX1119 exhibit a large safety window wherein the concentrations at which they target AML cells do not affect normal healthy cells such as liver epithelial cells or lung fibroblasts. To further establish BTX508 as a clinical candidate for AML, we performed in vivo efficacy studies in the MV-4-11 human AML xenograft model using athymic nude mice. A single daily dose of BTX508 results in a significant reduction in tumor volume. BTX508 exhibits significant oral bioavailability, thus making it a promising clinical candidate for treatment of AML as well as other potential hematological malignancies. With regard to BTX1119, we believe that incorporation of both the potent cytotoxic and immunomodulatory properties into a single molecule holds great therapeutic promise; however, to evaluate the synergistic potential of this combination will require use of a humanized mouse model - this work is ongoing. Disclosures No relevant conflicts of interest to declare.

Core Transcriptional Regulatory Circuitries in AML

Core transcriptional regulatory circuitries (CRCs) are tightly integrated networks of master transcription factors (TFs) that establish and maintain lineage-specific programs of gene expression. We hypothesized that divergent CRCs establish distinct subtypes of acute myeloid leukemia (AML). CRCs are defined as sets of master TFs that are marked by superenhancers (SEs) and bind to their own genes and those of the other core TFs, forming feed-forward auto-regulatory loops. We have performed large-scale H3K27ac ChIP-seq experiments to map the SE landscape in 20 AML cell lines, 3 normal hematopoietic tissues and 50 patient-derived xenograft (PDX) models of human AML. These experiments highlighted a core set of SE-marked, highly expressed TF genes shared by all the examined AML subtypes, corresponding to a putative pan-AML CRC. Importantly, a significant majority (&amp;gt;70%) of these transcription factors correspond to AML-specific genetic dependencies in the Project Achilles genome-scale RNAi and CRISPR-Cas9 screening efforts undertaken by colleagues at the Broad Institute, confirming the specific reliance of AML on these TFs for survival. We reasoned that CRCs can be predicted by integrating the epigenomic and functional dependency datasets. Indeed, intersecting SE-marked TF genes with preferential AML dependencies resulted in 32 candidate TFs. We have validated these TFs as AML dependencies in a low-throughput system with lentiviral delivery of Cas9 and specific gRNAs. ChiP-seq experiments with antibodies against 23 of these candidates (GATA2, PU1, IRF8, GSE1, GFI1, MEIS1, LYL1, CEBPA, MEF2D, MEF2C, IKZF1, ZEB2, FLI1, ETV6, ELF2, MAX, RUNX1, MYB, IRF2BP2, LMO2, SP1, ZMYND8, MYC) resulted in nearly 100% validation rate. They demonstrated that CRC TFs tend to co-occupy DNA and bind their own and each other's promoters and SEs, suggesting that CRC members function in higher-order chromatin complexes and establish reciprocal feed-forward regulatory loops. Analysis of TF co-binding revealed 237,636 unique binding sites, with most occupied by at least two CRC TFs. Specific combinatorial patterns of TF binding appear to be associated with promoters, enhancers and super-enhancers. Importantly, in addition to the pan-AML CRCs, highly specific dependencies restricted only to a subset of AML cell lines can be accurately predicted from examination of divergent (subtype-specific) AML CRCs, lending support to our hypothesis that context-specific vulnerabilities can be robustly inferred from a systematic study of TF circuits. Specifically, we identified MEF2D and IRF8 as TFs that are selectively marked by SEs in a subset of AML cell lines and PDXs, most notably in samples carrying an MLL rearrangement. At the same time, these genes are strong preferential dependencies in a subset of AML cell lines, most of which also carry an MLL fusion. This suggests specific roles of IRF8 and MEF2D in MLL-induced leukemogenesis. Interestingly, functional dependency scores for these two TFs show an extremely high degree of correlation, indicating tightly integrated functions. While IRF8 is a known regulator of macrophage/dendritic cell function, MEF2D has no recognized roles in hematopoiesis. We have validated MEF2D as a dependency in a low-throughput CRISPR-Cas9 drop out experiment in an MLL-rearranged cell line. At the same time, we observed no functional effect of MEF2D knock out in a human CD34+ cell colony forming assay. This confirms context-specific transcriptional addiction to MEF2D induced by an MLL fusion and suggests a potential "Achilles heel" for leukemia-specific therapy with little or no detrimental effects on normal hematopoiesis. In summary, our data allow us to draw the following conclusions: 1) Intersection of lineage-restricted gene dependencies with SE profiling permits highly specific discovery of CRCs. 2) Transcriptional control in AML is orchestrated by a large CRC of &amp;gt;30 essential TFs. 3) Divergent CRCs are diagnostic of cancer- and context-specific transcriptional addiction. 4) AML CRC is a highly integrated network of co-binding TFs that orchestrate both promoter- and enhancer-centric regulation. Figure Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents &amp; Royalties. Stegmaier:Novartis: Research Funding; Rigel Pharmaceuticals: Consultancy. Orkin:Syros: Consultancy; Novartis: Consultancy.

ETH-155008, a Novel Selective Dual Inhibitor of FLT3 and CDK4/6 in Preclinical Treatment of Acute Myeloid Leukemia

Fms-like tyrosine kinase 3 (FLT3) is part of a family of receptor tyrosine kinases (RTKs), and a molecular therapeutic target of acute myeloid leukemia (AML). There are three FTL3 inhibitors, midostaurin, gilteritinib, and quizartinib have been approved for clinical treatment of AML. We have evaluated the inhibition of cell proliferation in vitro and antitumor activity in vivo of ETH-155008, a novel triple inhibitor of FLT3, Pim-3 and CDK4/6. ETH-155008 was found to have a strong inhibitory activity against key target enzymes (FLT3 IC50 at 0.5nM; Flt3 -Itd IC50 at 0.6 nM; CDK4/D1 IC50 at 0.8nM, CDK6/D1 IC50 at 1.4nM), which were more potent than the tested positive controls gilteritinib, quizartinib, and AMG-925. ETH-155008 showed to have highly potent antiproliferative activities against cultured human acute myeloid leukemia cells (MV4-11 and Molm-13) with IC50 at 4.1nM and 12 nM, respectively, and showed remarkable antitumor efficacy in vivo. The in vivo antitumor activity of ETH-155008 was evaluated and compared with midostaurin and gilteritinib in the treatment of the two models with mutant forms in TKD and gatekeeper region, namely BaF3 FLT3-ITD-F691L and BaF3 FLT3-ITD-D835Y, respectively. Treatment with ETH-155008 (40 mg/kg/daily x 18, oral) significantly delayed tumor growth and resulted in a smaller mean tumor size (tumor growth inhibition (TGI) of 86% (p&lt;0.001 compared with the control group) in the BaF3 FLT3-ITD-F691L model, and cured all of the tumor-bearing mice in the BaF3 FLT3-ITD-D835Y model. The antitumor activity of ETH-155008 was comparable to gilteritinib, but much better than midostaurin, the latter showed only mild antitumor activity with ~25% of TGI in the two FLT3 mutant models. We also tested the therapeutic efficacy of ETH-155008 in the human AML xenograft model MV4-11, ETH-155008 cured all of the tumor-bearing mice (mean tumor size at time of treatment 126 mm3), at a dose of 40mg/kg/daily for 14 days, that showed very little toxicity (only 2% body weight loss) which were well below the maximum tolerated dose. ETH-155008 represents a novel dual inhibitor that may yet fulfill the promise of effective AML therapy through dual targeting of FLT3 and CDK4/6. These preclinical results supported the safety and efficacy observed in the preclinical studies and worthy of further clinical evaluation. Disclosures Liu: Euthare LLC: Other: founder. Zhang:shengke therapeutics: Other: cofounder. Yu:shengke Therapeutics: Other: co-founder.

Abstract 3833: Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC

Abstract Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel targets for the therapy of various malignant diseases. Also LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we have characterized a novel LSD1 inhibitor and assessed its potential as a novel therapy for AML and SCLC patients. Materials and Methods: Novel LSD1 inhibitor, HM97211 and its derivatives were designed and synthesized as the active biologic inhibitory compound against the AML and SCLC cells. Biochemical enzyme assay was performed to identify enzyme selectivity of HM97211 derivatives. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM97211 derivatives in AML and SCLC cell lines. In vivo anti-tumor activity was evaluated in AML and SCLC cell xenograft mice models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action. Results: HM97211 derivatives showed higher selectivity toward LSD1 compared to other FAD-utilizing enzyme as well as other epigenetic enzymes. HM97211 derivatives alter expression of various genes and induce differentiation and apoptosis, resulting in growth suppression in a panel of AML and SCLC cells. Moreover, HM97211 derivatives significantly induced programmed cell death in AML and SCLC cells. Oral administration of HM97211 derivatives inhibited tumor growth of human AML and SCLC xenograft models as a single agent at doses exhibiting significant. Efficacy of HM97211 derivatives was further evaluated in combination treatment with standard chemotherapies in human AML and SCLC xenograft model. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211 derivatives, can be a strong therapeutic agent for AML and SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy. Citation Format: InHwan Bae, WonJeoun Kim, JiSook Kim, JiYoung Song, JungSoo Nam, Moonsub Lee, Jooyun Byun, Hyeongki Kim, TaeHun Song, Seokhyun Hong, KyuHang Lee, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3833.

Small Molecular Modulators of Histone Demethylases Selectively Inhibits Growth of Hematopoietic Malignancies

Background: About 10% of acute leukemia (AL) patients harbor MLL-r(earrangements). MLLr acute myeloid leukemia (AML) mainly occurs in young-to-middle-aged adults whereas MLLr acute lymphoblastic leukemia (ALL) mainly occurs in patients younger than 1 year at diagnosis. AML with MLL fusion to MLLT3 via t(9;11)(p22;q23) predicts intermediate prognosis whereas MLL fusion to other partners predicts adverse prognosis. By contrast, in infants with ALL MLLr invariably confers poor prognosis. Much efforts have been made to identify and target proteins required for initiation and maintenance of MLLr AL, with an aim to improve the prognosis of this aggressive AL subtype. Multiple writers, erasers, and readers of histone post-translational modifications (PTMs) have been identified to be fundamental for the initiation and maintenance of MLLr AL. Small molecular inhibitors of some of these chromatin-associated proteins have been identified, such as EPZ004777 against DOT1L, JQ1 and I-BET151 against BRD4, and so on which are also under clinical trials for AL treatment. Among histone modification erasers essential for MLLr AL, JMJD1C and KDM4C that share Jumonji catalytic domain are fundamental for MLLr AL maintenance. Histone H3 lysine 9 (H3K9) demethylase JMJD1C is one of the most promising MLLr AL targets. Multiple independent studies identified JMJD1C as required for MLLr AML, RUNX1(AML1)/RUNX1T1(ETO) AML and even chronic myeloid leukemia and lymphoma cells but not normal hematopoiesis. KDM4C Is essential for Initiation and maintenance of MLLr AL transcriptional profiling of which is dependent on KDM4C. Moreover, pharmacological inhibition of KDM4C blocks leukemia development in syngeneic mouse model and human AML xenograft model. Although a large number of special inhibitors of histone demethylases have been developed, no special inhibitors against KDM3 family member like JMJD1C have been reported.Results: Here we focused on Jumonji domain that is responsible for enzymatic activities of histone demethylases for identifying potential small molecule modulators of histone demethylases. We selected Jumonji domain of histone H3 lysine (H3K9) demethylase JMJD1C with KDM4C as reference to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components through virtual screening method. Although identified independently from each other, compound #4 and #12 both share a common structural backbone and surface plasmon resonance analysis showed that #4 and #12 bind to JMJD1C, KDM3 family member KDM3B, and KDM4 family member KDM4C with modest affinity. In vivo demethylation assay showed that #4 induces global increase of H3K9 methylation. In vitro demethylation assay showed that #4 is able to reverse H3K9 demethylation conferred by KDM3B and KDM4C. We thus named #4 and #12 as JI-4 and JI-12 (JI, Jumonji inhibitor). Cell proliferation and colony formation assays showed that JI-4 and JI-12 predominantly kill MLLr AL. To increase evidence, multiple similar compounds to JI-4 and JI-12 were tested for cell proliferation repression and JI-16 was found to show superior killing activities against hematopoietic malignant cells compared to JI-4 and JI-12. Mechanistically, JI-16 not only induces apoptosis but also differentiation of MLLr AL cells. Transcriptome analysis and quantitative PCR (QPCR) showed that JI-16 induced gene expression profiling is especially enriched in gene sets involved in metabolism.Conclusion: To sum up, we identified potential pan-inhibitors of the Jumonji domain of histone demethylases. Binding in-vivo is followed by selective killing of MLLr AL cells.Disclosures. No relevant conflicts of interest to declare. DisclosuresNo relevant conflicts of interest to declare.

Chloroquine Derivative Lys05 Overcomes Hypoxia-Induced Chemoresistance in Acute Myeloid Leukemia through Metabolic Disruption

Background: Despite initial responses to standard chemotherapy, most patients with acute myeloid leukemia (AML) relapse and have poor prognoses after subsequent therapy. The hypoxic bone marrow (BM) microenvironment is hypothesized to contribute to clinical resistance through the sheltering of AML blasts and leukemia stem cells (LSCs), allowing them to evade chemotherapy and reinitiate the disease. We have previously shown that AML cell lines cultured under prolonged hypoxia (1% O2) can upregulate autophagy and become resistant to cytarabine (AraC). Furthermore, the inhibition of autophagosome turnover with either Bafilomycin A1 (Baf A1) or chloroquine (CQ) can re-sensitize AML cells to AraC. Additionally, Baf A1 therapy effectively eradicated in vivo functional LSCs in a primary human AML xenotransplantation mouse model. However, clinical development of BafA1 and CQ has been limited by poor drug pharmokinetics. Lys05 is a novel water-soluble CQ derivative which exhibits 10 fold more potent autophagy inhibitory properties than CQ in vitro and is well tolerated in mouse models. Here, we tested the effects of Lys05 on human AML growth, chemoresistance under hypoxia, and in vivo leukemia burden in a human AML xenograft model. We also explored the effects of autophagosome accumulation on cellular metabolism as a new mechanism for targeting chemoresistant AML blasts and LSCs.Methods: Human AML cell line MOLM13 and CD34 positive AML patient samples were cultured under hypoxia (1% O2) or normoxia (21% O2) with cytarabine (AraC) and late-stage autophagy inhibitor Lys05 alone and in combination. After 72 hours of drug exposure, apoptosis was measured using Annexin V/propidium iodide flow cytometry. Metabolic assays were performed using a Seahorse XFe96 analyzer to measure oxygen consumption and extracellular acidification rates using the Mitochondrial or Glycolytic Stress Tests on AML cells treated with late-stage autophagy inhibitors for 48 hours. Mitochondrial mass was measured following staining with MITO-ID Green Detection Kit using flow cytometry. Human xenograft models were performed using NSG mice inoculated with MOLM13 BLIV, a luciferase-expressing human AML cell line, and treated with either vehicle or Lys05 (20 mg/kg 4 days on/3 off for 4 weeks). Tumor burden was assessed using bioluminescence and analyzed with IVIS Living Image Software.Results: Treatment of AML MOLM13 cells with Lys05 in vitro resulted in a dose-dependent decrease in cell proliferation and increased apoptosis. Lys05 treatment further displayed similar results to BafA1 and CQ, enhancing the anti-leukemic effects of AraC and overcoming hypoxia-induced chemoresistance. Seahorse XFe analysis revealed that BafA1 inhibited both basal and maximal mitochondrial respiration under both normoxic and hypoxic conditions at 24 hours. CQ and Lys05, however, had no effect on oxidative phosphorylation under normoxia, yet significantly decreased mitochondrial function under hypoxia. Interestingly, reduced function did not coincide with a decrease in mitochondrial mass, but rather increased mitochondrial mass. We also observed decreases in glycolytic function after treatment with late-stage autophagy inhibitors under hypoxia suggesting these cells may be undergoing an energy crisis. Treatment of leukemia-bearing mice with Lys05 trended toward decreased tumor burden compared to vehicle. Studies optimizing Lys05 monotherapy and testing combination treatment with AraC are underway.Conclusions: Our research shows that CQ derivative Lys05 exhibits significant anti-leukemic activity against human AML cells and restores chemosensitivity to cytarabine under hypoxia, further supporting our hypothesis that autophagosome accumulation can overcome hypoxia-induced chemoresistance. We also demonstrate that Lys05 may effectively inhibit leukemia progression in vivo. Our studies further reveal metabolic disruption through the accumulation of autophagosomes as a mechanism for restoring chemosensitivity of AML cells under hypoxia and targeting LSCs. These results establish late-stage autophagy inhibitors, specifically Lys05, as a promising new treatment approach for resistant AML and supports further study of these drugs for treatment of minimal residual disease. [Display omitted] DisclosuresGuzman:Cellectis: Research Funding. Wang:Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Consultancy; Jazz: Speakers Bureau.

Human Acute Myeloid Leukemia Inhibits Normal Erythroid Differentiation through the Paracrine Effects of IL-6

Acute Myeloid Leukemia (AML) is an aggressive cancer resulting in severe cytopenias related to bone marrow (BM) failure. The common assumption for AML-induced BM failure is overcrowding due to clonal expansion of immature myeloid blasts, leading to failure of normal hematopoiesis. However, in a cohort of 293 AML patients, we found that disease burden (% of blasts determined on diagnostic BM aspirate) did not predict severity of cytopenia (hemoglobin rs=-0.053; p=0.49; WBC rs=-0.030, p=0.70; platelet rs=0.091, p=0.026), strongly arguing against simple crowding as the main mechanism underlying AML-induced BM failure. Thus, the goal of our study is to identify novel mechanism(s) associated with AML-induced BM failure, potentially enabling development of new therapies to improve AML management and reverse morbidity.Conventional xenograft models of human AML do not typically exhibit cytopenias, making them unsuitable to study AML-induced BM failure. We speculated that in the mouse, increased splenic extramedullary hematopoiesis compensated for failed intramedullary hematopoiesis due to AML. To test this hypothesis, we performed surgical splenectomy on NSG mice prior to their transplantation with human AML. Strikingly, splenectomized NSG mice engrafted with primary human AML at a 30-70% disease burden (n=8 for primary AML samples, n=5-10 for each group of splenectomized NSG mice) developed leukopenia and severe anemia compared to sham-operated AML-engrafted controls. AML-engrafted splenectomized NSG mice showed early mortality compared to AML-engrafted NSG mice with intact spleens (10.1 weeks vs. 34.2 weeks p<0.0001). Thus, our splenectomized murine xenograft model uniquely recapitulates human disease and allows for novel characterization of AML-induced BM failure.Utilizing our model, splenectomized NSG mice engrafted with human AML demonstrated depletion of normal hematopoietic progenitors (HSPCs) including HSCs (11.1 fold p<0.0001), MPPs (8.90 fold, p<0.001)), CMPs (11.4 fold p<0.0001), MEPs (1.76 fold p=0.0005) and GMPs (5.07 fold p<0.0001). Focusing on anemia, AML also depleted erythroid progenitors including proerythroblasts (1.39 fold p=0.04), normoblasts (4.96 fold p<0.0001), late normoblasts (10.0 fold p<0.0001), and reticulocytes (4.34 fold p=0.007). The relative preservation of proerythroblasts and depletion of normoblasts indicated that AML blasts impart a specific in vivo differentiation blockade in erythroid differentiation.To explore mechanisms by which AML blasts inhibit normal HSPCs, we generated conditioned media (CM) from patient-derived AML blasts, and found that AML-CM suppressed BFU-E colony formation from normal HSPCs (3.1-5.1 fold). Furthermore, in a direct co-culture system with AML blasts and CD34+ HSPCs, AML blasts inhibited erythroid differentiation from the CFU-E to normoblast stage by 81%. Removing CD34+ cells from the AML co-culture allowed cells to resume differentiation to the proerythroblast stage. These experiments demonstrate that AML imparts a differentiation blockade along the MEP-proerythroblast axis in a cell non-autonomous, reversible fashion.Using cytokine array analysis, we identified elevated IL-6 levels in AML sample-derived CM (n=10, 2841±766.4 pg/ml, 7.80 fold increase, p=0.03) compared to CD34+-derived CM (n=5, 364±36.0 pg/ml). Increased IL-6 was also found in BM aspirates from human AML engrafted splenectomized NSG mice (715±125pg/ml, p=0.001) compared to mice engrafted with normal CD34+ HSPCs (undetectable). Thus, we hypothesized that IL-6 produced by AML blasts acts as a paracrine factor to suppress erythropoiesis. Consistent with this hypothesis, an IL-6 neutralizing antibody reversed the inhibition of BFU-E formation imparted by AML-CM. Furthermore, the addition of recombinant IL-6 to liquid cultures of erythroid differentiation resulted in a 23% reduction in proerythroblasts.Together, our data suggests that (1) overcrowding is not the primary mechanism resulting in BM failure in AML; (2) AML blasts play a previously unrecognized role in imparting a differentiation blockade along the MEP-proerythroblast axis, resulting in progressive anemia; and (3) this differentiation blockade is at least partially attributable to IL-6 secreted from AML blasts as a paracrine factor. DisclosuresNo relevant conflicts of interest to declare.

Abstract 951: Direct small-molecule BAX activation in acute myeloid leukemia

Abstract Resistance to apoptosis is a hallmark of human cancer. The BCL-2 protein family includes both pro- and anti-apoptotic proteins and has a central role in regulating mitochondrial apoptosis. Cancer cells most frequently ensure their survival and resistance to treatments by overexpression of anti-apoptotic BCL-2 proteins to maintain BAX and other pro-apoptotic members suppressed. Pro-apoptotic BAX is the cardinal executioner BCL-2-family member that, upon conformational activation and oligomerization at the mitochondrial outer membrane (MOM), causes permeabilization of the MOM and release of mitochondrial factors, e.g., cytochrome c, that activate the caspase cascade of apoptosis. Interestingly, the vast majority of cancer cells contain functional BAX in an inactive conformation or suppressed by anti-apoptotic proteins; mutations or alterations in BAX that may cause its inactivation to occur at a low frequency. Therefore, we hypothesized that induction of apoptosis by direct activation of BAX offers the possibility of a new anticancer strategy. Using a structure-based drug design approach we developed BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site (trigger site) and induces conformational changes to BAX, resulting in BAX mitochondrial translocation, oligomerization and mitochondrial dysfunction (Reyna et al., Cancer Cell 2017). BTSA1 induces activation of cytosolic BAX, leading to prompt and robust mitochondrial apoptosis in acute myeloid leukemia (AML) cell lines. The efficacy of BTSA1 is regulated by the availability of anti-apoptotic BCL-2 proteins to inhibit activated BAX and therefore, higher levels of cytosolic BAX monomer correlated with higher efficacy of BAX-mediated mitochondrial dysfunction. BTSA1-induced BAX activation promotes effectively apoptosis in primary AML samples and has significant antitumor activity in human AML xenograft models, increasing host survival. BTSA1 is orally bioavailable with excellent pharmacokinetics at therapeutically effective doses; it does not show any detectable toxicity in the hematopoietic system or other tissues. Importantly, BTSA1 is also effective in inducing apoptosis in leukemic stem cell-enriched fractions (CD34+CD38−) while sparing healthy counterparts. Consistent with their sensitivity to direct BAX activation, AML blasts and highly purified stem and progenitor cell populations from AML patients when compared to healthy counterparts displayed higher expression of BAX. Overall, our data provide proof of concept for BAX as a druggable target and demonstrate the therapeutic potential of direct BAX activation by BTSA1 as an effective treatment strategy in AML. Citation Format: Denis Reyna, Thomas Garner, Andrea Lopez, Felix Kopp, Gaurav Choudhary, Ashwin Sridharan, Swathi-Rao Narayanagari, Kelly Mitchell, Baoxia Dong, Boris Bartholdy, Loren Walensky, Amit Verma, Ulrich Steidl, Evripidis Gavathiotis. Direct small-molecule BAX activation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 951.

Abstract 484: Tumor suppressor miRNA-101 modulates leukemogenesis by targeting the EZH2/Wnt/β-catenin signaling pathways

Abstract The survival rate of acute myeloid leukemia (AML) patients undergoing standard chemotherapy remains poor due to disease relapse. The main cause of relapse is the failure of chemotherapeutic drugs to eradicate self-renewing leukemic stem cells (LSCs) (Cancers 2017;9:74-97). Because of their intrinsic drug resistance, LSCs are extremely difficult to target by single-gene therapies. MicroRNAs (miRNAs), a class of small noncoding RNAs capable of regulating multiple target genes, represent a promising therapeutic target for elimination of LSCs. While miRNA-101 (miR-101) has been identified as a tumor suppressor in several types of cancers including prostate (Science 2008;322:1695-99) and liver cancer (Cancer Res 2009;69:2623-29), its expression and functional role in AML remains unknown. In this study, we uncover that miR-101 plays a crucial role in leukemogenesis through modulation of key epigenetic and signaling pathways activated in LSCs of mixed-lineage leukemia (MLL)-rearranged AML. Our data showed that overexpression of miR-101 in murine pre-LSCs inhibited cell proliferation in vitro and delayed leukemogenesis in vivo, confirming a tumor-suppressor function of miR-101 in MLL-AF9-induced AML, a highly aggressive leukemia. To assess the therapeutic value of miR-101, we next examined the antitumor function of miR-101 in a xenograft model of human MLL-AF9 AML. Briefly, miR-101 was overexpressed in human MLL-AF9 AML cell line, MOLM-13, that was then transplanted into NOD-scid IL2Rgammanull (NSG) mice followed by in vivo bioluminescence imaging to monitor engraftment of human leukemic cells. Our result revealed a significantly lower engraftment in the xenograft mouse model carrying miR-101-overexpressing leukemic cells compared to control mice. This suggests that inhibition of tumor suppressor miR-101 is essential in the initiation of an aggressive form of human AML. To investigate the potential mechanism by which miR-101 impairs leukemogenesis, we examined key oncogenic pathways regulated by miR-101 by performing luciferase assays and Western blotting. Our data showed that miR-101 inhibited leukemogenesis via directly targeting Enhancer of zeste homolog 2 (EZH2), a crucial epigenetic regulator in leukemia maintenance, as well as key components of Wnt/β-catenin signaling, an oncogenic pathway necessary for LSC self-renewal in AML. Furthermore, immunofluorescence analysis revealed a significant reduction in active β-catenin in the nucleus of miR-101-overexpressing leukemic cells, indicating a direct regulation of miR-101 in β-catenin activity. Collectively, these findings reveal a novel role for miR-101 in leukemia progression that may be used to develop a targeted therapy for AML treatment. Citation Format: Estrella Gonzales-Aloy, Dylan G. Grebert-Wade, Jenny Y. Wang. Tumor suppressor miRNA-101 modulates leukemogenesis by targeting the EZH2/Wnt/β-catenin signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 484.

Abstract 1876: INCB052793, a JAK1 selective inhibitor, is highly efficacious in PDX and xenograft models of acute myeloid leukemia (AML) expressing elevated endogenous pSTAT3/pSTAT5

Abstract Acute myeloid leukemia (AML) is characterized by infiltration of abnormally differentiated, clonal and highly proliferative cells of the hematopoietic system that acquire successive genomic alterations. AML is the most common acute leukemia in adults. Current therapies are of limited utility and involve a combination of cytarabine and anthracycline based regimens with allogeneic stem cell transplantation for eligible candidates, but there is an urgent need to improve therapies for AML. JAK/STAT pathway dysregulation plays a role in the pathogenesis of AML and the JAK2 V617F mutation is present in only a small percentage of these patients. Studies were conducted to evaluate the in vitro and in vivo activities of INCB052793, a highly JAK1-selective inhibitor having 100-fold selectivity for JAK1 over JAK2 in cell lines, xenograft and PDX models of human AML having elevated endogenous pSTAT3 and or pSTAT5 activation. In vitro, INCB052793 effectively inhibited p-Stat3 and/or p-Stat5 phosphorylation MV411, Molm 16 and Molm 13 cell lines and caused marked reductions in p-Akt and c-Myc levels in MV411 and Molm16 cells. Given these observations, oral administration of INCB052793 was evaluated at doses of 3-30 mg/kg twice daily in MOLM-16 xenografts and FLT3-ITD AML xenograft models, MV-4-11 and Molm-13, in SCID mice. INCB052793 administration significantly inhibited tumor growth in MOLM-16 xenografts in a dose dependent manner and resulted in complete downregulation of p-Stat3 and p-Stat5 levels in MOLM-16 tumors. Similarly, INCB052793 administration was highly effective in inhibiting tumor growth in FLT3-ITD AML models, MV-4-11 and Molm-13. Administration of INCB052793 in a systemic PDX model of AML with elevated endogenous levels of p-Stat3 and p-Stat5 resulted in amelioration of disease severity and demonstrated a significant effect on median survival in leukemic SCID mice. All dosing regimens of INCB052793 in both xenograft and PDX models were well tolerated. Since azacitidine and cytarabine are standards of care for the treatment of AML, the efficacy of INCB052793 was benchmarked against optimal dosing regimens of these agents. In the AML xenograft models evaluated, INCB052793 was comparably or more efficacious in reducing tumor burden than azacitidine and cytarabine. The combination of INCB052793 with cytarabine showed superior efficacy in comparison to single agents in the MOLM-16 xenograft model, and combinatorial studies are in progress in additional AML models. These findings suggest the therapeutic potential of INCB052793 as a single agent and in combination with standard of care chemotherapeutic regimens for the management of AML. Citation Format: Ashish Juvekar, Sindy Condon, Xiaoming Wen, Bruce Ruggeri, Peggy Scherle, Reid Huber, Yunlong Li, Wenqing Yao, Song Mei, Deepak Bhasin, Maria Mancini. INCB052793, a JAK1 selective inhibitor, is highly efficacious in PDX and xenograft models of acute myeloid leukemia (AML) expressing elevated endogenous pSTAT3/pSTAT5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1876.